U.S. patent number 11,441,143 [Application Number 17/119,473] was granted by the patent office on 2022-09-13 for enzyme-carrier complex.
This patent grant is currently assigned to Korea Univercity Research and Business Foundation. The grantee listed for this patent is Korea University Research and Business Foundation. Invention is credited to Jungbae Kim, Tae Hee Kim.
United States Patent |
11,441,143 |
Kim , et al. |
September 13, 2022 |
Enzyme-carrier complex
Abstract
The present invention relates to an enzyme-carrier complex, and
more particularly to the adsorption and stabilization of an enzyme
on the surface of a carrier, and an enzyme-carrier complex with
secured enzyme stability so that an enzyme immobilized by a
hydrophobic interaction exhibits long-term enzymatic activity.
Inventors: |
Kim; Jungbae (Seoul,
KR), Kim; Tae Hee (Seoul, KR) |
Applicant: |
Name |
City |
State |
Country |
Type |
Korea University Research and Business Foundation |
Seoul |
N/A |
KR |
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Assignee: |
Korea Univercity Research and
Business Foundation (Seoul, KR)
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Family
ID: |
1000006557108 |
Appl.
No.: |
17/119,473 |
Filed: |
December 11, 2020 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20210115432 A1 |
Apr 22, 2021 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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PCT/KR2020/001530 |
Jan 31, 2020 |
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Foreign Application Priority Data
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Jan 31, 2019 [KR] |
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10-2019-0012434 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N
9/96 (20130101); C12Y 305/01004 (20130101); C12N
11/14 (20130101) |
Current International
Class: |
C12N
11/14 (20060101); C12N 9/96 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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105836731 |
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Aug 2016 |
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CN |
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106636058 |
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May 2017 |
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CN |
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106676092 |
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May 2017 |
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CN |
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10-1995-0008685 |
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Apr 1995 |
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KR |
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10-0351887 |
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Nov 2002 |
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KR |
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10-2011-0128134 |
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Nov 2011 |
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KR |
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10-2016-0092652 |
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Aug 2016 |
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KR |
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79/00875 |
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Nov 1979 |
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WO |
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Other References
Basso et al., "Industrial applications of immobilized enzymes--A
review", Molecular Catalysis 479 (2019) 110607 (Year: 2019). cited
by examiner .
U.S. Appl. No. 17/118 814, filed 2020. cited by examiner .
Byoung Chan Kim et al., "Fabrication of enzyme-based coatings on
intact multi-walled carbon nanotubes as highly effective electrodes
in biofuel cells", Scientific Reports, 2017, pp. 1-10, vol. 7,
40202. cited by applicant .
Tae Hee Kim et al., "Biocatalytic membrane with acylase stabilized
on intact carbon nanotubes for effective antifouling via quorum
quenching", Journal of Membrane Science, 2018, pp. 357-365, vol.
554. cited by applicant .
International Search Report for PCT/KR2020/001530, dated Jun. 19,
2020. cited by applicant.
|
Primary Examiner: Davis; Ruth A
Attorney, Agent or Firm: Sughrue Mion, PLLC
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation application of, and claims
priority from International Application No. PCT/KR2020/001530 filed
Jan. 31, 2020, claiming priority from Korean Patent Application No.
10-2019-0012434 filed Jan. 31, 2019, the entire disclosures of
which are incorporated herein by reference.
Claims
The invention claimed is:
1. An enzyme-carrier complex comprising: a hydrophobic carrier
which is a carbon nanotube; and one or more acylases adsorbed on a
surface of the hydrophobic carrier, wherein the enzyme-carrier
complex does not form aggregates in a dispersed state in water.
2. The enzyme-carrier complex of claim 1, wherein the hydrophobic
carrier further comprises a first functional group on a surface
thereof to induce a hydrophobic interaction with the enzyme.
3. The enzyme-carrier complex of claim 2, wherein the one or more
acylases further comprises a second functional group for inducing a
hydrophobic interaction with the first functional group.
4. The enzyme-carrier complex of claim 3, wherein the second
functional group for inducing a hydrophobic interaction comprises
one or more functional groups selected from the group consisting of
a halogenated alkyl group, an organosilicon group, an alkyl group,
a vinyl group, an allyl group, and an aryl group.
5. An electrode for a biofuel cell, the electrode comprising the
enzyme-carrier complex according to claim 1.
6. An electrode for a biosensor, the electrode comprising the
enzyme-carrier complex according to claim 1.
7. A carbon dioxide conversion system comprising the enzyme-carrier
complex according to claim 1.
8. An antifouling system comprising the enzyme-carrier complex
according to claim 1.
Description
TECHNICAL FIELD
The present invention relates to an enzyme-carrier complex, and
more particularly to the adsorption and stabilization of an enzyme
on the surface of a carrier, and an enzyme-carrier complex with
secured enzymatic stability so that an enzyme immobilized by a
hydrophobic interaction exhibits long-term enzymatic activity.
BACKGROUND ART
Enzymes have the characteristics of high ultra-precision,
specificity, selectivity, and high efficiency, and thus the use
thereof is expanding to various industries run by humans, and in
terms of function, enzymes not only have the general function of
catalyzing redox, transition, hydrolysis, desorption and addition,
isomerization, and synthesis reactions, but also have the
characteristic of catalyzing reactions under special conditions
such as high temperature, high pressure, and organic solvents, and
thus an industrially application range thereof is unlimited, and
accordingly, utilization thereof as a future industrial material is
very high.
However, enzymes are easily structurally denatured by external
environments such as heat and pH, and thus there is a problem that
application thereof is highly limited due to the instability of
enzymatic activity. In order to address this problem, recently,
research on gene recombination technology or enzyme immobilization
technology has been actively conducted to enhance the stability of
an enzyme.
With regard to the enzyme immobilization technology, a technique
for modifying the surface of a carrier with a functional group
capable of binding to an enzyme, and then immobilizing the enzyme
on the carrier through covalent bonding via the functional group is
actively being researched. When this method is used, stable binding
can be ensured, but there is a problem that the activity of an
enzyme to be immobilized is deteriorated or lost due to structural
denaturation of the enzyme occurring during a reaction for covalent
bonding. In addition, there is a disadvantage that an additional
functionalization process is required to modify the surface of a
carrier so as to have a functional group, and in such a
functionalization process, the intrinsic properties of a carrier
may be changed or deteriorated.
Therefore, there is an urgent need for an enzyme immobilization
technique which is able to immobilize an enzyme on a carrier while
minimizing the deterioration of enzymatic activity, maintain
long-term enzymatic activity, and minimize the deterioration of
intrinsic properties of the carrier due to modification.
DESCRIPTION OF EMBODIMENTS
Technical Problem
The present invention has been made in view of the above problems,
and it is an object of the present invention to provide an
enzyme-carrier complex in which an enzyme is immobilized on a
carrier while the deterioration of enzymatic activity is minimized,
that is able to secure enzyme stability so that long-term enzymatic
activity is exhibited, and that is able to minimize the
deterioration of intrinsic properties of a carrier.
Also, it is another object of the present invention is to provide
an enzyme-carrier complex capable of securing dispersibility in an
aqueous solution.
Technical Solution
According to an aspect of the present invention, there is provided
an enzyme-carrier complex including a hydrophobic carrier and an
enzyme adsorbed on a surface of the hydrophobic carrier.
According to one embodiment of the present invention, the enzyme
may include one or more enzymes selected from the group consisting
of acylase, trypsin, chymotrypsin, pepsin, lipases, glucose
oxidase, pyranose oxidase, horseradish peroxidase, thyroxinase,
carbonic anhydrase, formaldehyde dehydrogenase, formate
dehydrogenase, alcohol dehydrogenase, cholesterol dehydrogenase,
lactonase, proteases, peroxidases, aminopeptidases, phosphatases,
transaminases, serine-endopeptidase, cysteine-endopeptidase, and
metalloendopeptidases.
In addition, the hydrophobic carrier may include one or more
materials selected from the group consisting of carbon nanotubes,
fullerenes, graphene, porous carbon, polycarbonate, polyimide,
polystyrene, polydimethylsiloxane, and polyethylene
terephthalate.
In addition, the hydrophobic carrier may further include a first
functional group on a surface thereof to induce a hydrophobic
interaction with the enzyme.
In addition, the enzyme may further include a second functional
group for inducing a hydrophobic interaction with the first
functional group.
In this regard, the second functional group for inducing a
hydrophobic interaction may include one or more functional groups
selected from the group consisting of a halogenated alkyl group, an
organosilicon group, an alkyl group, a vinyl group, an allyl group,
and an aryl group.
In addition, the enzyme-carrier complex may be any one selected
from the group consisting of an acylase-carbon nanotube complex, a
trypsin-carbon nanotube complex, a lipase-carbon nanotube complex,
a glucose oxidase-carbon nanotube complex, a pyranose
oxidase-carbon nanotube complex, a horseradish peroxidase-carbon
nanotube complex, a tyrosinase-carbon nanotube complex, a carbonic
anhydrase-carbon nanotube complex, and a formaldehyde
dehydrogenase-carbon nanotube complex.
The present invention also provides an electrode for a biofuel cell
including the enzyme-carrier complex according to the present
invention.
The present invention also provides an electrode for a biosensor
including the enzyme-carrier complex according to the present
invention.
The present invention also provides a carbon dioxide conversion
system including the enzyme-carrier complex according to the
present invention.
The present invention also provides an anti-fouling system
including the enzyme-carrier complex according to the present
invention.
Advantageous Effects of Invention
An enzyme-carrier complex according to the present invention
enables an enzyme to be stably immobilized on a carrier without
denaturation of the active site of the enzyme, and after
immobilization, can exhibit long-term enzymatic activity. In
addition, even after the immobilization of an enzyme on a carrier,
deterioration of the intrinsic properties of the carrier can be
minimized, and thus it is advantageous in that the intrinsic
properties of the carrier are fully exhibited. Moreover, even when
the carrier has hydrophobic properties, it is possible to secure
dispersibility in an aqueous solution, and thus it is suitable for
use in various applications requiring long-term stable enzyme
performance. In addition, without using components having a
biocompatibility problem, such as a crosslinking agent, which have
been applied to an enzyme stabilization technique, the enzyme is
immobilized and stabilized, and thus use of the enzyme-carrier
complex can be expanded to the medical field where use of such
components is limited.
Accordingly, when the enzyme-carrier complex is used in the
immobilization of related enzymes, such as glucose oxidase and
pyranose oxidase, in order to manufacture glucose-based biofuel
cells and biosensors for measuring blood glucose, a very high
stabilization effect can be achieved. In addition, when the
enzyme-carrier complex is used in the immobilization of related
enzymes, such as carbonic anhydrase capable of converting carbon
dioxide into bicarbonate and formate dehydrogenase capable of
converting bicarbonate into formic acid, it is possible to stably
maintain activity for a long time without denaturation of an
enzyme, compared to conventional immobilization methods using a
crosslinking agent, and thus the enzyme-carrier complex can be used
as a catalyst material for a carbon dioxide conversion and
utilization system. In addition, when the enzyme-carrier complex is
used in the immobilization of related enzymes such as acylase which
is able to inhibit biofilm formation by decomposing a signaling
molecule that performs a quorum sensing function, the activity of
an enzyme can be stably maintained without denaturation thereof
compared to conventional immobilization methods using a
crosslinking agent and accordingly, the enzyme-carrier complex can
be used as a catalyst material in an antifouling system to suppress
the formation of a biofilm on the surface of a membrane. The
enzyme-carrier complex can also be used in various applications
such as electrochemical and pharmaceutical industries in addition
to the above-listed examples.
BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 is a view of an enzyme-carrier complex according to an
embodiment of the present invention.
FIGS. 2 and 3 are graphs for evaluating enzyme stability by
comparing an enzyme-carrier complex according to an embodiment of
the present invention with an enzyme in a free state and each of
enzyme-carrier complexes according to comparative examples.
FIG. 4 illustrates an image and view showing a state in which an
enzyme-carrier complex according to an embodiment of the present
invention is dispersed in water.
FIG. 5 illustrates an image and a view showing a state in which
carbon nanotubes used as a carrier in the complex of FIG. 4 are
dispersed alone in water.
BEST MODE
Hereinafter, embodiments of the present invention will be described
in detail in such a manner that the invention can be carried out by
one of ordinary skill in the art to which the present invention
pertains, without undue difficulty. The present invention may be
embodied in many different forms and is not limited by embodiments
set forth herein.
Referring to FIG. 1, an enzyme-carrier complex according to the
present invention includes a hydrophobic carrier and an enzyme
adsorbed on a surface of the hydrophobic carrier. In one
embodiment, the absorption may occur by a hydrophobic interaction
between a hydrophobic moiety of the enzyme and the surface of the
hydrophobic carrier.
Any hydrophobic carrier may be used without limitation as long as
it is an insoluble material that is commonly referred to as
exhibiting hydrophobicity in the art. In the present invention, the
hydrophobic carrier refers to a carrier capable of having a van der
Waals interaction or a pi-pi interaction, among non-covalent bonds,
with the surface of the enzyme on the surface thereof that can
contact the surface of the enzyme. In addition, any hydrophobic
carrier may be used without limitation as long as it is a material
as defined above, and may include, for example, one or more
materials selected from the group consisting of carbon nanotubes,
fullerenes, graphene, porous carbon such as nanoporous carbon or
activated carbon, polycarbonate, polyimide, polystyrene,
polydimethylsiloxane, and polyethylene terephthalate.
The shape of the hydrophobic carrier is not limited, and may be,
for example, one or more shapes selected from the group consisting
of spherical, plate, rod, tubular, and amorphous shapes. In
addition, the hydrophobic carrier may further include nano-sized
pores in the surface thereof, but the present invention is not
limited thereto.
In addition, the size of the hydrophobic carrier may vary from
nanoscale to microscale, but the present invention is not
particularly limited thereto.
In addition, the hydrophobic carrier may further include, on the
surface thereof, a first functional group to induce a hydrophobic
interaction with the enzyme or to further enhance the hydrophobic
interaction. The hydrophobic interaction minimizes or prevents an
effect on the steric structure of an enzyme compared to covalent
bonding, and thus is advantageous in that it can prevent or
minimize problems such as denaturation of the steric structure of
an enzyme and deterioration or loss of enzymatic activity, which
occur according to strong binding affinity caused when an enzyme is
immobilized on a support via conventional covalent bonding or when
enzymes are crosslinked via covalent bonding. In this regard, the
first functional group included in the hydrophobic carrier is not
limited as long as it is a functional group capable of inducing a
hydrophobic interaction, and may include, for example, one or more
functional groups selected from the group consisting of a
halogenated alkyl group, an organosilicon group, an alkyl group, a
vinyl group, an allyl group, and an aryl group.
In addition, the first functional group may be introduced onto the
surface of the hydrophobic carrier through a known technique, but
the present invention is not particularly limited thereto.
As the enzyme, any known enzyme may be employed without limitation,
and may include, for example, one or more enzymes selected from the
group consisting of acylase, trypsin, chymotrypsin, pepsin,
lipases, glucose oxidase, pyranose oxidase, horseradish peroxidase,
thyroxinase, carbonic anhydrase, formaldehyde dehydrogenase,
formate dehydrogenase, alcohol dehydrogenase, cholesterol
dehydrogenase, lactonase, proteases, peroxidases, aminopeptidases,
phosphatases, transaminases, serine-endopeptidase,
cysteine-endopeptidase, and metalloendopeptidases. More preferably,
the enzyme may include one or more selected from the group
consisting of acylase, trypsin, lipases, glucose oxidase, pyranose
oxidase, horseradish peroxidase, thyroxinase, carbonic anhydrase,
and formaldehyde dehydrogenase, and even more preferably, the
enzyme may be acylase.
In addition, according to one embodiment of the present invention,
the enzyme may further include a second functional group for
enhancing a hydrophobic interaction with the hydrophobic carrier.
The second functional group may function to minimize the effect on
the steric structure of the enzyme while minimizing dissociation of
the enzyme from the hydrophobic carrier, and stably exhibit
enzymatic activity. The second functional group may include one or
more functional groups selected from the group consisting of a
halogenated alkyl group, an organosilicon group, an alkyl group, a
vinyl group, an allyl group, and an aryl group. In addition, the
second functional group may be selected from those that are the
same as or different from the first functional group that may be
included in the hydrophobic carrier, but the present invention is
not particularly limited thereto.
According to one embodiment of the present invention, the enzyme
and the hydrophobic carrier in the above-described enzyme-carrier
complex may be any one combination selected from an acylase-carbon
nanotube complex, a trypsin-carbon nanotube complex, a
lipase-carbon nanotube complex, a glucose oxidase-carbon nanotube
complex, a pyranose oxidase-carbon nanotube complex, a horseradish
peroxidase-carbon nanotube complex, a tyrosinase-carbon nanotube
complex, a carbonic anhydrase-carbon nanotube complex, and a
formaldehyde dehydrogenase-carbon nanotube complex, and due to an
enhanced interaction between the enzyme and the surface of the
carrier in such a combination, the enzyme may be stably
immobilized, and there is an advantage that long-term enzymatic
activity may be exhibited.
Mode of Invention
Hereinafter, the present invention will be described in detail with
reference to the following examples. However, these examples are
not intended to limit the scope of the present invention.
Example 1>--Preparation of Enzyme-Carrier Complex Using Acylase
and Carbon Nanotubes (ADS-AC/CNTs)
A carbon nanotube (CNT) solution (8 mg/mL) and an acylase (AC)
solution (40 mg/mL), prepared using phosphate-buffered saline (100
mM, pH 7.0) as a solvent, were mixed in the same volume ratio,
followed by stirring at 200 rpm for 1 hour to thereby adsorb
acylase onto the surfaces of carbon nanotubes. Subsequently,
acylase unattached to the carbon nanotubes was removed using
phosphate-buffered saline, followed by stirring with Tris buffer
(100 mM, pH 7.4) at 200 rpm for 30 minutes, causing unreacted
functional groups to be capped. Thereafter, the prepared
enzyme-carrier complex was centrifuged to remove the supernatant,
washed with phosphate-buffered saline, and then stored at 4.degree.
C.
<Comparative Example 1>--Free Acylase Solution (Free AC)
An acylase solution dissolved in phosphate-buffered saline (100 mM,
pH 7.0) was prepared.
<Experimental Example 1> Measurement of Enzymatic Activity
and Stability of Enzyme-Carrier Complex Using Acylase and Carbon
Nanotubes
As an enzyme, acylase (AC), which decomposes bacterial
quorum-sensing signaling material, was used. AC activity was
measured using fluorescence emitted as a result of reaction of
L-methionine produced by hydrolysis of N-acetyl-L-methionine with
o-phthalaldehyde (OPA).
Enzyme stability was evaluated by measuring a decrease in enzymatic
activity while continuing to stir at 200 rpm after a sample was
dispersed in phosphate-buffered saline. Specifically, relative
activity with respect to initial activity of the enzyme-carrier
complex of Example 1 using acylase and carbon nanotubes and the
free acylase solution of Comparative Example 1 was evaluated for
200 days, and the results thereof are shown in FIG. 2.
As can be confirmed from FIG. 2, the enzyme in the case of
Comparative Example 1 was non-activated within 19 days, whereas the
case of Example 1 maintained a relative activity of 20% for 200
days. Specifically, the case of Example 1 exhibited a relative
activity reduced to 23% for the first 40 days, and then stably
maintained a relative activity of 23% until 200 days had
elapsed.
Experimental Example 2
The enzymatic activity and stability of each of the enzyme-carrier
complexes according to Example 1 and Comparative Example 2 were
measured in the same manner as in Experimental Example 1, and the
results thereof are shown in FIG. 3.
As can be confirmed from FIG. 3, the case of Example 1 maintained a
relative activity of 20% for 200 days, whereas the case of
Comparative Example 2 did not exhibit enzymatic activity after
being immobilized.
<Comparative Example 3>--Carbon Nanotube (CNT) Solution
A solution in which carbon nanotubes (8 mg/mL) were added to
phosphate-buffered saline (100 mM, pH 7.0) was prepared.
Experimental Example 3
Each of the enzyme-carrier complex according to Example 1 and the
carbon nanotubes according to Comparative Example 3 was added to a
water-containing container and stirred at 200 rpm for 1 hour, and
then each container was left on a table for 1 minute, followed by
photographing, and acquired images are shown in FIGS. 4 and 5.
As illustrated in FIG. 4, it can be confirmed that the
enzyme-carrier complex of Example 1, in which acylase is adsorbed
on surfaces of the carbon nanotubes, is uniformly dispersed in
water, from which it can be seen that the enzyme-carrier complexes
are not aggregated.
In contrast, as illustrated in FIG. 5, it can be confirmed that,
when carbon nanotubes alone are dispersed in water, most settle on
the bottom, and even when carbon nanotubes do not settle, the
carbon nanotubes are present as grains aggregated therebetween.
* * * * *